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Keywords:

  • Drosophila;
  • enhancer analysis;
  • hematopoiesis;
  • plasmatocyte;
  • Serpent

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. METHODS
  7. Acknowledgements
  8. LITERATURE CITED

Eater is a transmembrane protein that mediates phagocytosis in Drosophila. eater was identified in a microarray analysis of genes downregulated in S2 cells, in which Serpent had been knocked down by RNAi. The gene was shown to be expressed predominantly in plasmatocytes after embryonic development. We have extensively analyzed the transcriptional enhancer controlling eater expression with the following findings: the enhancer reproduces the plasmatocyte expression pattern of the gene as verified by anti-P1 antibody staining and a 526-basepair DNA region is active in lymph gland and hemolymph plasmatocytes. This DNA contains several GATA elements that serve as putative-binding sites for Serpent. Site-directed mutagenesis of two of these GATA sites abolishes eater expression in both lymph gland and hemolymph plasmatocytes. This suggests that Serpent regulates eater expression by binding these GATA sites, which was confirmed by gel shift analysis. These analyses allowed us to use eater-Gal4 to force plasmatocyte to lamellocyte differentiation. genesis 50:41–49, 2012. © 2011 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. METHODS
  7. Acknowledgements
  8. LITERATURE CITED

Drosophila has emerged as an important model organism for the study of hematopoiesis. Although much simpler than mammalian hematopoiesis, many developmental aspects of Drosophila hematopoiesis remain evolutionarily similar. These similarities include conserved signaling molecules and transcription factors, the myeloid cell lineage, and the biphasic nature of hematopoiesis (Evans et al.,2003). Because of these similarities and advancement in genetic techniques, Drosophila can be manipulated to study the effect that specific gene perturbation may have on hematopoiesis.

Hematopoiesis occurs in two spatiotemporal waves. In the embryo, hemocytes form in the cephalic mesoderm and remain in circulation throughout larval development (Holz et al.,2003). These cells proliferate until there exists between 6,000 and 8,000 hemocytes by the end of the third larval instar (Sorrentino et al.,2007). The second wave occurs in a specific organ, termed the lymph gland, during larval stages. The lymph gland is composed of three domains: medullary zone (MZ), cortical zone (CZ), and posterior signaling center (PSC). The MZ is composed of hematopoietic stem-like cells called prohemocytes, and the CZ consists of mature hemocytes. The PSC, located at the posterior tip of the lymph gland, expresses signaling molecules such as Serrate, Hedgehog, and Unpaired to maintain prohemocytes in the MZ (Jung et al.,2005; Krzemien et al.,2007; Mandal et al.,2007; Tokusumi et al., 2010). During the third instar stage, differentiation of prohemocytes in the lymph gland occurs in the border region of the MZ and CZ and mature hemocytes form in the CZ, while the prohemocytes are still maintained inside the MZ (Jung et al.,2005; Krzemien et al.,2010). At the onset of metamorphosis, the lymph gland ruptures, releasing a massive amount of mature hemocytes into circulation (Lanot et al.,2001).

Drosophila have three known types of blood cells derived from a common precursor: plasmatocytes, crystal cells, and lamellocytes. The most abundant cell type, composing 95% of cells in the hemolymph, is the plasmatocyte, similar to macrophages in mammals. They are small round cells that are responsible for phagocytosis of microbes, debris, apoptotic cells, and remodeling of tissues during metamorphosis (Rizki and Rizki, 1984; Tepass et al.,1994). They can also secrete antimicrobial peptides to aid in immunity (Lanot et al.,2001). There are two distinct populations of plasmatocytes, circulating and sessile. Crystal cells comprise about 5% of hemocytes in the Drosophila hemolymph and are known to have crystalline inclusions of Prophenoloxidase, a proenzyme that is processed to form an enzyme required for melanization during wound healing. Lamellocytes are large, flat adherent cells that differentiate in response to infection with parasitic wasp eggs or when genetically induced. The function of a lamellocyte is to encapsulate objects that are too large for plasmatocytes to phagocytize.

A GATA factor, Serpent (Srp), is expressed in all types of hematocytes and is essential for hematopoiesis. Crystal cells are regulated by a RUNX factor, Lozenge (Lz). Lz can interact with Srp to direct crystal cell differentiation (Lebestky et al.,2000). Drosophila Friend of GATA homologue, U-shaped (Ush) interacts with Srp to limit crystal cell fate (Fossett et al.,2003; Waltzer et al.,2003). Furthermore, Notch (N) pathway enhances crystal cell differentiation (Duvic et al.,2002; Lebestky et al.,2003). Lamellocytes are regulated by the JAK/STAT, JNK, N, Wingless, and Toll pathways (Duvic et al.,2002; Luo et al.,1995; Sorrentino et al.,2002; Tokusumi et al.,2009a; Zettervall et al.,2004). Glial cells missing (GCM) and glial cells missing 2 (GCM2) regulate plasmatocyte differentiation in embryonic stages (Lebestky et al.,2000), but these genes are not essential for larval plasmatocyte differentiation, because they are not expressed in lymph glands (Avet-Rochex et al.,2010; Evans et al.,2003). Thus, it remains unknown what factors direct plasmatocyte differentiation within lymph glands.

As the plasmatocyte is the main hemocyte type involved in innate immunity, there are many transmembrane proteins important in antigen recognition and ultimately phagocytosis. Eater is a type I transmembrane protein with several EGF-like repeats that plays an important role in this antigen recognition and phagocytosis (Kocks et al.,2005). Eater was discovered in an RNAi knockdown of Srp in Drosophila cultured S2 cells and subsequent microarray analysis. Eater is expressed in mature plasmatocytes and therefore worthwhile to study to determine the molecular mechanism of plasmatocyte differentiation during larval stages. Previously, we reported a 1.7 kb upstream region of eater can direct plasmatocyte expression (Sorrentino et al.,2007; Tokusumi et al.,2009b). In this work, we further delimit this region to 580 bp and a Srp-binding cluster in this DNA is essential for this restricted pattern of eater expression.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. METHODS
  7. Acknowledgements
  8. LITERATURE CITED

Spatial and Temporal Expression Pattern of Eater Enhancer-Driven Reporter Genes

Previously, we identified a 1767 bp eater enhancer region that can direct blood cell expression of the GFP reporter gene (Fig. 1a; Sorrentino et al.,2007; Tokusumi et al.,2009b). The eater enhancer-driven GFP expression was detected in sessile hemocytes, residing under the cuticle, as well as the lymph glands (Fig. 1b). In lymph glands, eater-GFP was strongly expressed in the CZ, which consists of mature hemocytes (Fig. 1c). eater expression is found predominantly in cells of the CZ as indicated by a lack of colocalization of eater-DsRed with the MZ marker TepIV-Gal4>mCD8::GFP (Fig. 1d). In circulation, most hemocytes were GFP-positive, because plasmatocytes constitute ∼95% of mature hemocytes (Fig. 1e). Further experimentation suggested the full-length fragment conferred expression in all circulating plasmatocytes, as shown by anti-P1 plasmatocyte specific antibody staining in hemolymph samples (Fig. 1e,f).

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Figure 1. Analysis of the eater enhancer recapitulates larval plasmatocyte expression of the gene in both the lymph gland and hemolymph. (a) A 1767 base pair DNA upstream of eater possesses enhancer activity. (b) As seen through the larval cuticle, eater-GFP is present in all lobes of the late third instar larval lymph gland (inside box) as well as bands of sessile and circulating plasmatocytes. (c) Transgene activity within a dissected lymph gland from eater-GFP larva indicates that in the primary lobes, eater is highly expressed in cells of the CZ, where plasmatocytes reside. Expression is also present in the secondary and tertiary lymph gland lobes of late third instar larvae, likely in prohemocytes. (d) eater expression is abundant in cells of the CZ, as indicated by lack of colocalization of eater-DsRed and MZ marker TepIV-Gal4 > UAS-mCD8::GFP. (e) eater-GFP is present in circulating plasmatocytes from hemocyte samples of larvae with the eater-GFP transgene. (f) The same hemolymph sample was also stained with P1 plasmatocyte-specific antibody.

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Srp Controls the Eater Enhancer Through a GATA-Binding Motif

To determine the important cis-elements in this enhancer, subsequent truncations were generated. Both eater F9 and eater F10, which delete 437 bp and 1 kb of enhancer distal DNA, conferred plasmatocyte GFP expression. A similar result was seen with eater F11, which shortened the full-length DNA to a 580 bp region (Fig 2a). When truncated from the 5′ end to 100 bp (eater F13), the result was a complete loss of GFP expression in both the lymph gland and circulating plasmatocytes. This indicated one or more cis-elements reside within that 480 bp region between eater F11 and eater F13. Within this fragment, there are two distinct GATA cis-element clusters of four and two sites (Fig. 2a). Because eater was identified by GATA factor Srp RNAi knockdown, and Srp is expressed in the lymph gland and plasmatocytes, we postulated that Srp was a regulator of eater expression. Therefore, we decided to analyze the importance of these GATA factor clusters. When all four upstream GATA sites were mutated in the F11 fragment, GFP expression was still detected in both the lymph gland and circulating plasmatocytes (Fig. 2a, eater F11mGATA 1D). However, mutation of the two proximal elements induced a complete loss of GFP expression in both lymph glands and circulating hemocytes (Fig. 2a, eater F11mGATA 1B). This suggested that one or both GATA sites play a crucial role in eater plasmatocyte expression.

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Figure 2. Molecular dissection of the minimal eater enhancer to identify its regulatory elements. (a) Schematic of truncations, chosen by distance between GATA elements. Whole larvae were screened for eater-GFP in the lymph gland, and hemolymph was obtained to identify GFP in circulating plasmatocytes. Two individual clusters of GATA elements were simultaneously mutated (1D and 2B) to determine the cis-element(s) to which Srp may bind. (b) Double-stranded probes were designed to the two proxmial GATA binding elements within the eater enhancer region. (c) An electrophoretic mobility gel shift assay was performed with a 32P-labeled probe designed to the final GATA site in the regulatory region (lane 1). Srp protein was generated using in vitro translation and incubated with the 32P-labeled probe causing a shift (lane 2). A 20-fold excess of unlabeled wild-type probe was incubated with 32P-labeled probe and protein abolishing the shift (lane 3). A 200-fold excess of mutant unlabeled probe was incubated with 32P-labeled probe and protein causing a shift in the gel (lane 4).

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Srp is known as a crucial GATA factor regulating Drosophila hematopoiesis (Evans et al.,2003; Sorrentino et al.,2005). Therefore, a competition gel shift assay was performed to determine if and where Srp binds within the eater enhancer region. Through our enhancer analysis, it was clear that at least one of the final two GATA sites was responsible for the expression of eater. To identify which of the GATA sites binds Srp, we used two different oligonucleotides designed to the distal and proximal GATA sites (Fig. 2b). The gel shift assay with these two DNAs revealed that Srp bound to only the proximal GATA element within the eater enhancer region (Fig. 2c, lane 2). Both isoforms of the Srp protein were used to perform the gel shift assay, with no significant difference detected between them. To confirm this binding precisely, a competition binding assay was performed. A 20-fold excess of unlabeled wild-type DNA ablated the complex of Srp protein and labeled DNA (Fig. 2c, lane 3). In addition, to ensure the specificity of this binding, 200-fold excess of the oligonucleotide, including the mutated GATA-binding site, was incubated with labeled wild-type DNA and Srp protein. This mutated DNA did not ablate the protein-DNA complex (Fig. 2c, lane 4). Thus, we concluded that Srp can specifically bind to the proximal GATA site to regulate eater expression in plasmatocytes.

Spatial and Temporal Regulation of Eater Expression

To determine the spatial and temporal expression patterns of eater driven-reporter genes, we observed lymph glands and hemocytes, along with other mature hemocyte markers, during different developmental stages. At 72 h after egg lay (AEL), eater-DsRed was detected in lymph glands, whereas a crystal cell marker, lz-Gal4, was not observed at this time (Fig. 3a). The detection of lz-Gal4;UAS-GFP began at 96 h (data not shown). Expression of both genes remains detectable in the lymph gland through 120 h of development (Fig. 3b). Both eater-DsRed and lz-Gal4 were expressed in circulating hemocytes earlier than in the lymph gland, after only 48 h of development. Interestingly, a subset of eater-DsRed and lz-Gal4;UAS-GFP co-labeled cells was detected at this early time point (Fig. 3e). Both genes continued to be expressed throughout 120 h of development (Fig. 3f). Contrary to the earlier stages of development, eater-DsRed and lz-Gal4;UAS-GFP tend not to co-label as often at this later stage, although some co-labeling has been observed (data not shown).

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Figure 3. Spatial and temporal expression of eater-GFP and eater-Gal4 in dissected lymph glands and larval hemocyte samples. (a) eater-DsRed expression is observed in the CZ of dissected lymph glands of lz-Gal4 > UAS-GFP; eater-DsRed larvae at 72 h of development. (b) lz-Gal4; UAS-GFP and eater-DsRed are expressed through 120 h of development in dissected lymph glands. (c) eater-Gal4 UAS-2XeYFP expression is observed in the CZ of dissected lymph glands of eater-Gal4 UAS-2xeYFP; BcF6-CFP; MSNF9mo-mCherry after 96 h of development. (d) At 120 h of development, larvae with the genotype eater-Gal4 UAS-2xeYFP; BcF6-CFP, MSNF9mo-mCherry continue to express eater-YFP in both dissected lymph glands and also express MSNF9mo and Bc. (e) eater-DsRed as well as lz-Gal4 > UAS-GFP are expressed in circulating hemocytes after 48 h of development in lz-Gal4 > UAS-GFP; eater-DsRed larvae. (f) Both lz-Gal4 > UAS-GFP and eater-DsRed are expressed through 120 h of development in circulating hemocytes. (g) eater-Gal4; UAS-2XeYFP is expressed after 48 h of development in circulating plasmatocytes of eater-Gal4 UAS-2xeYFP; BcF6-CFP; MSNF9mo-mCherry larvae. (h) At 120 h of development, larvae with the genotype eater-Gal4 UAS-2xeYFP; BcF6-CFP; MSNF9mo-mCherry continue to express eater-YFP in circulating hemocytes, with few cells expressing MSNF9 or Bc.

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We also determined eater-Gal4 expression precisely. Lymph glands from eater-Gal4; UAS-2xeYFP BcF6-CFP MSNF9mo-mCherry were also dissected at the same time points and kept at 25°C (Tokusumi et al.,2009b). This line was used to compare the expression patterns of other hemocyte markers, as Bc marks mature crystal cells and MSN marks lamellocytes. In the lymph gland, eater-Gal4 began to be expressed in the cortical zone at 96 h AEL, 24 h later than eater-DsRed, while there was no expression of Bc or MSNF9 (Fig. 3c). Expression of eater-Gal4 remained through 120 h of development, when both Bc-CFP and MSNF9mo-mCherry appeared (Fig. 3d). Similar to eater-DsRed, eater-Gal4 expresses earlier in circulation than in the lymph gland. After only 48 h of development, eater-Gal;UAS-2xeYFP was detected in circulating plasmatocytes, while Bc and MSNF9 were not yet expressed (Fig. 3g). eater-Gal4 expression was maintained in circulation through 120 h of development, at which point Bc expression appeared (Fig. 3h). Also, at this later time point, MSNF9 was expressed in lamellocytes, which appeared naturally (data not shown). It has been noted that eater-Gal4;UAS-2xeYFP can mark some mature crystal cells, but these are few in number.

Plasmatocyte to Lamellocyte Differentiation

Understanding the expression patterns of eater reporters in mature plasmatocytes has allowed us to assess the potential of plasmatocyte to lamellocyte differentiation. In healthy larva, there are few, if any, lamellocytes in circulation or in the lymph gland. A previous study has shown that srp is involved in lamellocyte differentiation (Sorrentino et al.,2007). Thus, we tested misexpression of srp by eater-Gal4. Hemolymph was obtained from eater-Gal4;UAS-GFP > UAS-SrpC larvae (Fig. 4a) and stained with DAPI and L1, a lamellocyte specific antibody (Fig. 4b). Some of the hemocytes expressed both GFP driven by eater-Gal4 and the anti-L1 antigen. Similar results were obtained when SrpNC was also misexpressed using the eater-Gal4 driver. These results suggest that these cells might have the properties of plasmatocytes as well as lamellocytes, although only a small subset of L1 positive cells was also eater-GFP positive. Furthermore, eater-GFP expression in lamellocytes was weaker than in plasmatocytes. Together, this was suggestive that these cells were in a transition phase between plasmatocytes and lamellocytes.

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Figure 4. Forced expression of lamellocyte inducing factors using eater-Gal4 causes lamellocyte formation in hemocytes from third instar larvae. (a, b) eater-Gal4; UAS-GFP > UAS-SrpC hemolymph samples were stained with an anti-L1 lamellocyte-specific antibody and DAPI. (c, d) Hemolymph from eater-Gal4 UAS-2xeYFP; BcF6-CFP; MSNF9mo-mCherry crossed with UAS-vinculin RNAi larvae was analyzed. (e, f) Samples of eater-Gal4 UAS-2xeYFP; BcF6-CFP; MSNF9mo-mCherry crossed with UAS-EcRDN larval hemolymph were collected and observed.

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Recently, during an in vivo RNAi screen, we found vinculin RNAi driven by eater-Gal4 can induce lamellocytes, a subset of which express both eater-GFP (Fig. 4c) and MSNF9-mCherry, a lamellocyte marker (Fig. 4d). Another result of our RNAi screen led us to examine ecdysone receptor mutants. Hemolymph from eater-Gal4; UAS-2xeYFP BcF6-CFP MSNF9mo-mCherry > UAS-EcRDN larvae was also collected, with similar results to the vinculin knockdown, where a subset of these cells express both eater (Fig. 4e) and MSNF9 (Fig. 4f). In this sample, it is evident that eater also labels plasmatocytes as normal. The co-labeling of subsets of hemocytes with eater-GFP or eater-Gal4 and lamellocyte markers indicates that at least a subset of lamellocytes can be derived from mature plasmatocytes.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. METHODS
  7. Acknowledgements
  8. LITERATURE CITED

Specificity of Eater Reporter Is Controlled by Srp

The discovery of eater and its expression in blood cells by Kocks et al. (2005) led us to examine the enhancer region upstream of the gene to determine how it is transcriptionally regulated. We generated an enhancer-GFP construct that directs GFP expression in cells where Eater is expressed. The success of the eater-GFP enhancer allowed for the generation of eater-Gal4 flies, which is significant, because genes can be selectively expressed in plasmatocytes. Importantly, both eater-GFP and eater-Gal4 are expressed in all circulating plasmatocytes as well as the cortical zone of the lymph gland, the same staining pattern as anti-P1 plasmatocyte-specific antibody. Although eater-GFP and eater-Gal4 are detected in a small population of crystal cells, they are expressed mainly in plasmatocytes. To date, eater-GFP is one of few in vivo, live-cell plasmatocyte-active markers, and therefore will have many useful applications.

Kocks et al. (2005) first identified eater in an RNAi knockdown of Srp and subsequent microarray, which led us to hypothesize eater was regulated by Srp. We designed specific truncations of the eater regulatory region between clusters of GATA sites to identify the cis-element to which Srp may bind. We refined the enhancer to a 480 base-pair region in which eater expression was lost. Following the knockout of the two GATA sites nearest the eater gene, its expression was abolished. This finding argues for the importance of Srp in plasmatocyte differentiation. Furthermore, a gel shift assay was performed to identify the specificity of Srp binding to the enhancer region. Srp specifically binds the proximal GATA site within the enhancer, as it shows competition by the wild-type probe, but the mutant probe does not compete. These results suggest that Srp is at least, in part, responsible for the regulation of eater gene expression.

Different Expression Patterns of Lymph Glands and Circulating Plasmatocytes

eater is expressed strongly in cells of the lymph gland CZ and plasmatocytes as anticipated. Interestingly, eater-GFP and eater-Gal4 are expressed in plasmatocytes in circulation before being expressed in the lymph gland. It is possible that eater is expressed earlier in circulation than in the lymph gland, because circulating hemocytes are derived from the embryonic wave of hematopoiesis, while the lymph gland does not disperse until metamorphosis. The plasmatocytes that reside in the lymph gland do not need to mature until a late larval period, causing the delay in eater expression compared to circulating plasmatocytes. In circulation, it has been observed that lz and plasmatocyte-specific markers can both label the same cell (Lebestky et al.,2000). Using the eater enhancer to drive GFP, we also determined that there is a subset of eater-positive cells that co-localize with crystal cell markers lz and Bc. It was observed early in the development of lz-Gal4;eaterDsRed larvae that the amount of colocalization was greater than what was observed late in larval development. It is known that Lz is expressed in immature as well as mature crystal cells. It is possible that prohemocytes may express both lz and eater before terminal differentiation into either a crystal cell or a plasmatocyte. Another hypothesis is there is a threshold of lz that, once passed, causes the prohemocyte to become a crystal cell. Similarly, there is a possibility that a threshold of GCM/GCM2 may be reached causing a plasmatocyte fate. If these factors are in equilibrium, and no threshold has been met, the cell may express both eater and lz. However, later in development, some colocalization of eater and lz was observed, although it was less often than earlier in development. Furthermore, the eater expression in these cells was vastly reduced when compared with plasmatocytes in the same field suggesting that the cells are in a cell fate transition.

Lamellocytes Are Derived from Plasmatocytes

It has been suggested that lamellocytes are derived from sessile hemocytes in the posterior of the animal (Márkus et al.,2009). To further investigate the origin of lamellocytes, circulating hemocytes of various genotypes were observed. eater-Gal4 was used to drive lamellocyte-inducing genes in plasmatocytes. Hemolymph was collected from these animals and co-labeling was observed between lamellocyte markers and eater-GFP, suggesting that the lamellocyte was derived from a circulating plasmatocyte. There may be a subset of plasmatocytes that are able to differentiate into lamellocytes upon genetic induction or wasp parasitization, as not all lamellocytes were eater-positive. It was also observed that eater-GFP had a decreased expression level in lamellocytes as opposed to plasmatocytes. This decrease in eater may be caused by the loss of plasmatocyte identity and the gain of lamellocyte identity. Taken together, these findings are supportive of the existence of a plasmatocyte to lamellocyte differentiation program (see Fig. 5).

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Figure 5. Factors controlling cell fate decisions during Drosophila hematopoiesis. Prohemocytes give rise to all three known types of Drosophila hemocytes. Crystal cells are specified by N signaling as well as an interaction between the Lz and Srp transcription factors. This cell fate is inhibited by an interaction between Ush and the SrpNC isoform. Gcm and Gcm2 regulate the differentiation of plasmatocytes, the macrophages within the hemolymph. In nature, lamellocytes are rare, although they become abundant in response to Leptopilina wasp parasitization. Lamellocytes can also be induced by genetic perturbation of specific genetic programs. Similar to the crystal cell lineage, the lamellocyte fate is suppressed by Ush. Lamellocytes can also be derived from a subpopulation of plasmatocytes.

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METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. METHODS
  7. Acknowledgements
  8. LITERATURE CITED

Drosophila Strains and Crosses

w1118, y1 w67c23, lz-Gal4;UAS-EGFP, He-Gal4, UAS-EcRDN, and UAS-GFP stocks were obtained from the Bloomington Stock Center. UAS-Vinculin RNAi was obtained from the Vienna Drosophila RNAi Center. UAS-srpC and eater-Gal4 2xeYFP; BCF6-CFP, MSNF9mo-mCherry have been previously described (Fossett et al.,2003; Tokusumi et al.,2009b).

Generation of Transgenic Drosophila for Eater Enhancer Analysis

To identify cis-elements in the eater plasmatocyte-specific enhancer, we used PCR to generate a deletion series of the eater upstream 1767 base-pair DNA fragment (Sorrentino et al.,2007; Tokusumi et al.,2009b). Mutations in potential cis-element-binding sites were introduced into the 580 bp DNA fragment with the QuickChange site-directed mutagenesis kit (Stratagene). Following sequence confirmation, the mutated DNA was subcloned into the pH Stinger vector. All enhancer analyses were conducted with y1 w67c23 or w1118 larva, using standard Drosophila transformation procedures (Rubin and Spradling,1982). Five lines of each truncation were established and analyzed.

Hemocyte Analysis and Immunostaining

To identify GFP-positive Drosophila larvae, wandering third instar larvae were viewed using a Zeiss Stereo Lumar fluorescence stereomicroscope. To analyze circulating plasmatocytes, larval hemolymph samples were obtained in 10 μl of PBS on a glass slide and allowed to settle for 30 min in a humidity chamber. Live cells were observed with a Zeiss Axioplan 2 microscope. For DAPI and phalloidin staining, cells were fixed in 4% paraformaldehyde followed by incubation in 0.1% Triton X-100-PBS including Alexa Fluor 594-conjugated phalloidin and DAPI. Images were obtained by Zeiss Axioplan 2 microscope.

For immunostaining, samples were fixed in 4% paraformaldehyde and incubated in 0.1% Triton X-100-PBS plus 5% goat serum and primary antibody. After washing several times, samples were incubated with AlexaFluor 555-conjugated anti-mouse Ig G antibody (1:200; Molecular Probes). Images were captured using a Zeiss Axioplan 2 microscope or a Nikon A1-R confocal microscope. For timed developmental stage analysis, egg-lays were performed at 25°C for 4 h, and hemolymph samples were obtained or lymph glands were dissected at the times indicated.

Electrophoretic Mobility Shift Assay

TNT Quick Coupled Transcription/Translation Systems kit (Promega) was used to generate Srp protein. pCMVTnT-SrpC was used as a template for this reaction (Muratoglu et al.,2006). Oligonucleotides designed as probes were end-labeled with 32P using T4 Polynucleotide Kinase (Roche) and purified using G-25 Sephadex Quick Spin Columns for Radiolabeled DNA (Roche). The gel shift assay-binding reactions were performed using 25 μl of mixture containing 12.5 μl binding buffer (40 mM Tris [pH 8.0], 200 mM KCl, 10 mM MgCl2, 0.4 mM EDTA, 200 μg/ml BSA, 20% glycerol, 2 mM DTT), 2 μl of 1 μg/μl poly(dI-dC), 2 μl of 10 nM ZnSO4, 4 μl of in vitro translated protein, and 32P-end-labeled oligonucleotides. For competition assays, reaction mixture including excess amounts of wild-type unlabeled or mutated DNA was incubated at room temperature for 20 min. Reactions were then separated on a 0.5× TBE nondenaturing polyacrylamide gel.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. METHODS
  7. Acknowledgements
  8. LITERATURE CITED

We are grateful for stocks obtained from the Bloomington Stock Center and the Vienna Drosophila RNAi Center. We are also thankful to I. Ando for providing antibodies and N. Fossett for plasmids. This work was facilitated through the use of the Notre Dame Integrated Imaging Facility.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. METHODS
  7. Acknowledgements
  8. LITERATURE CITED